Protein subcellular localization and functional studies in horticultural research: problems, solutions, and new approaches

© The Author(s) 2023. Published by Oxford University Press on behalf of Nanjing Agricultural University. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Horticulture Research, 2023, 10: uhac271

To ensure different biochemical reactions take place simultaneously, eukaryotic cells have evolved several membranebounded organelles, which coordinate their functions through proteins involved in signal transduction, vesicle trafficking, and membrane interactions [1]. Therefore, determining the subcellular localization of a protein is an essential step toward understanding its functions and activities. To do this, people normally co-express genes of interest with a f luorescent organelle marker using the N. Benthamiana (Nicotiana benthamiana) transient expression system and image it with confocal microscopy [2]. This method is commonly used in horticulture research, where generating stable transgenic plants is still challenging for most species. Although there are a few common standards for this operation, such as signals should not be over-saturated, controls should be performed to eliminate the possibility of signal cross-talk between multiple channels (bleed-through), and the microscope settings (e.g. laser output, pinhole) should be kept identical when comparing two independent experiments, things may still go wrong in some circumstances. Here, we have highlighted a few examples of inappropriate image acquisition that happened in various previous studies and provide our reasonings and solutions to these issues.
Despite the potential issues of using a heterogeneous expression system, images taken from inappropriate focal planes may produce further difficulties to interpret the correct protein localization. For example, in mature leaf cells, more than 90% of the volume is occupied by the vacuole, and all cytoplasmic localized organelles are pushed towards the cell cortical region (Fig. 1a) [3]. The distance between f luorescent protein-labelled structures within this confined space is close to the resolution limit of normal light microscopy. Therefore, it is not possible to distinguish colocalizing signals from the background if images are taken from the middle section of a cell. As shown in Fig. 1b, free GFP or RFP localizes to the cytoplasm, and their signals are distinct from most subcellular structures (e.g cytoskeleton, mitochondria). However, if images were taken from the middle section, it is then impossible to tell the differences, and the signal distribution analysis tends to indicate strong co-localizations in all tests ( Fig. 1c and d). This is problematic when studying proteins associated with cytoplasmic structures or localized to the cytosol, as their localization may become indistinguishable from the plasma membrane (PM) or cytosolic background. To solve this problem and achieve the best resolution of most organelles (except for the nucleus), we recommend that images should be taken from the cortical section of leaf epidermal cells, which is the cytoplasmic layer (approximately 1 μm thick) that is positioned in-between the vacuole membrane (tonoplast) and the plasma membrane (Fig. 1a).
Furthermore, over-expressing some membrane proteins destined for secretion (e.g. to the PM or vacuole) may get trapped in some intermediate compartment and produce false labelling. For example, PIP2A (plasma membrane intrinsic protein 2A) is an aquaporin protein that is commonly used as a PM marker [2]. However, when its expression level is high, PIP2A accumulates in the ER and cannot be transported to the PM effectively (Fig. 2a). A similar effect is also likely found with other secretory membrane proteins at are produced in the ER. Therefore, it is critical to make sure that the marker proteins are localized correctly at the start of an experiment, and using a lower optical density (OD 600 < 0.05) of Agrobacterium for infiltration may help (Fig. 2a). However, protein expression level varies from cell to cell, so it could be difficult for non-experienced researchers to determine cells with optimized protein expression and correct localization. To better overcome these problems, we have generated a collection of stable N. Benthamiana lines expressing FPs-tagged organelle marker [2]. These include the mitochondria marker, MT-RFP; the autophagosomes marker, GFP-ATG8a; the plasma membrane marker, PIP2A-CFP; the plastid marker, PT-RFP; the actin cytoskeleton marker, GFP-Lifeact; and the microtubule marker, RFP-TUA5. Fluorescent markers in these plants exhibit uniformed expression levels and correct subcellular localizations (Fig. 2b); therefore, they can be used for protein co-localization studies, producing more consistent results.
Additionally, these stable N. benthamiana plants can also be used to characterize protein function at the subcellular level. Here, we used the RFP-TUA5 lines to study the functions of different microtubule regulators as examples. It is known that microtubules play an important role in plant development and fruit shape formation [4]. Therefore, we cloned the katanin p60 catalytic subunit (SlKTN1) and SlMAP65-1 of tomato (Solanum lycopersicum) and studied their localization and roles in cortical microtubule rearrangement. Transient transformed GFP-SlKTN1 fusion was found in the cytoplasm (Fig. 2c). However, with the increase of SlKTN1 expression, the degree of microtubule depolymerization is also enhanced, confirming the SlKTN1 from tomato has a function to cut microtubules, as reported in Arabidopsis thaliana [5]. Similarly, the transgenic plants were infiltrated with a 35S:GFP-SlMAP65-1, the protein not only co-localized with RFP-TUA5 labelled microtubules (Fig. 2d), but also induced microtubule bundles formation, suggesting the SlMAP65-1 from tomato may have the capacity to cross-link microtubules [6]. Because stable microtubule marker plants were used in the study, it is confident to say that the effects of SlMAP65-1 or SlKTN1 on microtubule structure were caused by the function of the proteins, not the variation of microtubule markers that were expressed at a different level. In the same way, other transgenic marker lines can be used to study the localization and activity of a protein in different subcellular compartments.
These transgenic lines can also be used to study the effect of transcription factors on subcellular dynamics. For example, CYCD3;1 is a D-type cyclin that increases mitotic cycles and reduces endocycles [7,8]. When it was transiently transformed into the RFP-TUA5 line, characteristic microtubule arrays that represent phragmoplast or mitotic spindles were observed, suggesting the heterologous expression of CYCD3;1 in leaf epidermal cells is able to promote mitosis (Fig. 2e). The NAC domain transcription factor, VASCULAR-RELATED NAC-DOMAIN7 (VND7), is a key regulator of xylem vessel differentiation and secondary wall patterning [9]. When VND7 was transiently expressed in N. benthamiana leaf, it mimics the effect of xylem differentiation, where microtubules reorganized into thick bundles, which may act as the track to direct the movement of cellulose synthase complex and the deposition of cellulose microfibers (Fig. 2f). Similarly, other key transcription factors can be transiently transformed in these stable plants, and their functions from a cell biological perspective could be determined.
Taken together, determining the localization of a protein is the step forward in understanding the function of any particular molecular pathway. While the most promising results come from studies using the native species at the endogenous expression level, the transient expression system certainly provides a good alternative to rapid screening, especially useful for horticultural crops that are normally difficult for genetic transformation. Here, we have highlighted some common issues for bio-imaging and generated several stable transgenic N. benthamiana lines that can be used for studying protein localization and functions for further research.

Transient and stable transformation of N. benthamiana
Agrobacterium-mediated transient expression of N. benthamiana was performed as described before [10,11]. For stable transformation, surface-sterilized seeds of N. benthamiana plants were grown on Murashige and Skoog (MS) agar in a growth chamber with a 16 h : 8 h, light : dark regime under 25 degrees. Leaf segments of 4-week-old plants were inoculated with the Agrobacterium culture and co-cultivated for 2 days on MS media, supplemented with 6-Benzylaminopurine (6-BA; 1 mg l −1 ) and α-naphthalene acetic acid (NAA; 3 mg l −1 ). Putative transgenic shoots from the leaf explants were induced on the same medium supplemented with Carbenicillin salt (150 mg l −1 ) and Kanamycin Sulfate (50 mg l −1 ). Regenerated shoots were transferred to rooting media, including MS, with Carbenicillin salt (150 mg l −1 ). After rooting, the plants were transferred to soil and kept at 25 • C with a 16-h photoperiod.

Image acquisition and analysis
Images were taken using a laser scanning confocal microscope (Leica TCS SP8) in multi-track mode with line switching, and processed with Fiji-ImageJ (https://imagej.net/imagej-wiki-static/ Fiji). The signal distribution analysis of FPs-tagged organelle markers was performed using the Plot Profile tool. The FibrilTool plugin [13] was used to measure the overall anisotropy of microtubules in leaf epidermal cells (at least 30 cells were analysed) as described before [14].